Process for preparing penicillin



Patented Feb. 28, 1950 2,499,290 PROCESS FOR PREPARING PENICILLINQuentin a. Bartz, Detroit, Mich., assignor to Parke, Davis & Company,Detroit, Mich., a corporation of Michigan No Drawing. Application April22, 1944, Serial No. 532,355

13 Claims. (oi. 260402) w The inventionrelates to the preparation ofproducts derived from the mold, Penicillium notatum, which possess highactivity against pathogenic microorganisms.

It is known that the said species of mold, Penicillium notatum, producesa substance called penicillin having high activity againstcertainmicroorganisms. However, P nicillin is produced by Penicillium notatumonly in very small amount and associated with impurities which are verydiflicult to remove.

A further difliculty in the way of obtaining penicillin is that itsactivity is quickly lost in the presence of chemicals and solvents,required for its isolation and purification,- unless low temperatures,suitable pH values and other conditions are carefully controlled.

Example A 100 pound lot of wheat bran whole culture of Penicilliumnotatum approximately 3 days old, prepared for example as describedv incopending application of Katherine Yaw, Serial No. 466,669, filedNovember 23, 1942, now abandoned, is extracted with a total volume of'72 gallons of 95% denatured alcohol (formula No. 3A containing methanolas a denaturant). The alcoholic extract is concentrated in vacuo. Duringthe main part of the distillation, the temperature of the distillingliquid should be below 77 F. (25 C.). Toward the end of thedistillation, the temperature of the distilling liquid rises but must beprevented from going above about 98.6 F. (37 (1.). The concentrationprocess whereby alcohol is removed to leave an aqueous concentrate isdiscontinued when one small volume of concentrate upon being shaken withan equal volume of diethyl ether produces anv aqueous layer equal to orslightly greater than the original volume. At this point the volume isabout 4 gallons.

Variations in the moisture content of the bran culture cause a widevariation in the final volume of the aqueous concentrate. If too muchalcohol remains in it, the next step of extracting oily impurities willbe interfered with. On the other hand, if an attempt is made to bringthe concentrate to dryness, loss of activity occurs. Usually, alcoholcan be removed until the concentrate from 100 lbs. of culture comes downto a volume of about 2.4 to 4.8 gallons.

The pH of the concentrate is determined to be about 6.5. If the pH isbelow 5, it should be brought within the range of about 5 to '7 by thecareful addition of ice cold normal sodium hydroxide solution. Locallyhigh concentrations of alkali during this addition should be avoidedsince the activity is destroyed by an excess alkalinity.

The concentrate is chilled without delay in a refrigerator at about 23F. to 32 F. (5 C. to 0 C.). It is then allowed to stand at thistemperature for about 24 hours, at the end of which time the top layeris peeled or skimmed oil and discarded.

The cold aqueous layer that remains is treated without delay in order toextract oily impurities. Its pH is determined and found to be 6.2. Ifthe pH is below about 6, it should be adjusted to a pH of about 6.0 to7.6 by careful addition of ice cold normal sodium hydroxide solution,while avoiding destruction of activity by excessive local concentrationsof alkali. The aqueous layer is then extracted first with an equalvolume of peroxide free ether and then with one half its volume ofether. carded.

The ether extracted aqueous layer is cooled to less than 41 F. (5 C.)and one-half its volume of similarly cooled ethylene dichloride added.After the ethylene dichloride is added, the pH is adjusted to a valuewithin the range of about 1.9 to 3.0 by cautiously adding ice cold molarphosphoric acid (HaPO4). A brownish precipitate frequently forms duringthis acidification. The particle 'size of this precipitate apparentlydepends upon the rate of addition of the acid and the conditions ofstirring. The stirring should not be too vigorous or a troublesomeemulsion may be produced.

The mixture is allowed to separate into two layers, the lower ethylenedichloride layer drawn off and stored at once at low temperature (e. g.41 F.) The aqueous layer is washed twice with one-half its volume ofethylene dichloride and all of the ethylene dichloride extractscombinedand stored at 5 C. The. highly pigmented aqueous layer is discarded. g

I have found that the penicillin activity is quickly destroyed in a fewminutes at room temperature when the pH of the aqueous solution isaround pH 2. Hence, the importance of first adding ethylene dichloride,before increasing the acidity by means of aqueous acid, and thencarrying out the above ethylene dichloride extraction rapidly and at alow temperature. Once the extraction is accomplished, the activity ofthe penicillin is stable for several days.

About 28.4 grams of a suitable charcoal, such as the commerciallyproduced superheated steamactivated product'known as Norit, is added tothe cold ethylene dichloride extract for each gal- The ether extractsare dis-,

ion of extract. Too much Norit is not used, last the penicillin itselfbe absorbed. The mixture with Norit is stirred for about minutes,filtered, and the Norit washed twice with a small quantity of ethylenedichloride. For example, about 57 ml. of ethylene dichloride for each28.4 grams of Norit used can be employed for each washing. It ispreferred that the Norit be treated with hydrochloric acid, filtered andthen washed until chloride-free before using it to treat the ethylenedichloride solution of penicillin,

The ice cold filtrate and washings from the Norit treatment arecombined. They contain the penicillin. A gallon of this filtrate istaken and to it there is added 200 ml. of ice cold pmsenfree distilledwater. The mixture is stirred, cold normal sodium hydroxide solutionadded carefully, and the aqueous layer adjusted to pH about 5.5 to 1.0.Locally high concentrations of alkali and excess alkalinity are avoidedin this step in a order to keep'from destroying the penicillin activity.The aqueous'layer contains the sodium salt of penicillin.

The remainder of the ethylene dichloride filtratei'rom theNorittreatment is divided into five equal parts. The above mentioned aqueouslayer or extract containing the sodium salt of penicillin is used toextract one of these flve equal parts, after adjusting the aqueous layerin the same manner as before with cold pyrogen-free distilled water andalkali. The aqueous layer or extract thus obtained is then used toextract the four remaining parts in turn, alkali being added again eachtime to adjust the pH to about 6.5 to 7.0.

In the above removal of penicillin from the ethylene dichloride layerinto an aqueous layer, the water and ethylene dichloride are first mixedbefore adjusting to a higher pH. This results in some of the penicillinbeing extracted into the aqueous layer at pH about 3. which is" in theunstable range for penicillin. Hence, the neutralization must proceedwithout delay and care must be taken not to overstep the end point. Ihave found that the penicillin is fairly stable in aqueous solution atpH 8.3, but that the activity is rapidly destroyed even in the cold (41F. or 5 C.) at pH11.3.

which are not solvents for appreciable an. of penicillin at said pHvalues, such for example as ethylene dichloride, chloroform, amylacetate, etc. Also, instead of using ethylene dichloride in theextraction step prior to the treatment with activated charcoal, I mayuse other water-immiscible organic liquids which are solvents forpenicillin at a pH of 1.9 to 3.0, such for example as ether, chloroform,amyl acetate, etc.

My invention, in its broader aspects, is based on my discovery thatactivated charcoal may be used to selectively adsorb impurites away fromthe active penicillin,'thus providing a means for obtaining, on acommercial scale, a highly purified penicillin product withoutsubstantial loss of penicillin activity. a

What I claim-as my invention is: 1 1. A process for obtaining penicillinfrom an organicextract containing the same, which comprises treatingsaid organic extract with activated charcoal to adsorb impurities uponsaid charpenicillin from the charcoal, and washing the The dissolvedethylene dichloride is removed from the aqueous extract in high vacuumat 68 to 86 F. (20 to 30 C.). The solution is then assayed forpenicillin activity by the "cup method as described by Abraham et al.,Lancet 2:177, August 16, 1941, and later by Fiorey et al., Brit. J. Exp.Path. and Med. 23:120. June, 1942. The solution contains more than 2500units per ml., as a unit is defined in said publications. It is put intoampoules or vials and stored at 5 C. until ready for use.

The solutions are stable for weeks at 5 C. but it is preferred to putthe solution into open ampoules or vials. freeze the same and then drythe contents by evaporation at low temperature and pressure while in thefrozen state. The sealed ampoules of dried. material are then stable forlong periods and the penicillin product contained ing penicillin atleast partially freed from oils and at approximately neutral pH with anorganic solvent at a temperature not substantially'above 5 C. whilequickly thereafter adding aqueous mineral acid to adjust the pH of theaqueous phase to a value between about 1.9 and 3.0, separating anddiscarding the aqueous phase, treating the organic solvent extract withactivated charcoal to adsorb impurities upon said charcoal, andseparating the organic extract containing unadsorbed penicillin from thecharcoal and impurities.

5. A process for obtaining penicillin which comprises treating anaqueous solution containing penicillin-at least partially freed fromoils and at approximately neutral pH with ethylene dichloride at atemperature not substantially above 5 C. while quickly thereafter addingaqueous mineral acid to adjust the pH of the aqueous phase to a valuebetween about 1.9 and 3.0, separating and discarding the aqueous phase.treating the ethylene dichloride extract with activated charcoal toadsorb impurities upon said charcoal, and separating the ethylenedichloride extract containing unadsorbed penicillin from the charcoaland impurities.

6. A process for obtaining penicillin which comprises treating anaqueous solution containing penicillin at least partially freed fromoils and at approximately neutral pH with ethylene dichloride at atemperature not substantially above 5 C. while quickly thereafter addingaqueous phosphoric acid to adjust the pH of the aqueous phase to a valuebetween about 1.9

and 3.0, separating and discarding-the aqueous phase, treating theethylene dichloride'extract with activated charcoal to adsorb impuritiesupon said charcoal, and separating the ethylene dichloride extractcontaining unadsorbedpenicilli'n J from the charcoal and impurities.

7. A process for obtaining penicillin from a slightly acidic aqueousextract ofa penicillinproducing mold culture, which comprises chillingsaid aqueous extract to produce solid impurities therein, separatingsaidimpurities, extracting an organic solvent to the ether-extracted aqueouseral acid to mixture to adjust the pH of the aqueous phase to a valuebetween about 1.9 and 3.0, separating and discarding the aqueous phase,treating the organic solvent extract with activated charcoal to adsorbimpurities upon said charcoal, and separating the organic extractcontaining'unadsorbed penicillin from'the charcoal and impurities.- I

8. A-proces's for obtaining penicillin from a slightly acidic aqueousextract of a penicillinproducing mold culture, which comprises chilling.said aqueous-extract to produce :solid impurities therein, separatingsaid impurities, extracting tivated charcoal to adsorb impurities uponsaid charcoal, and separating the organic 1 extract containingunadsorbed penicillin from the charcoal and impurities.

9. A process for obtaining slightly acidic aqueous extract of apenicillinproducing mold culture, which comprises chilling saidv aqueousextract to produce solid impurities therein, separating said impurities,extracting further impurities from the aqueous residue at approximatelyneutral pH with ether, adding ethylene dichloride to the ether-extractedaqueous residue, quickly thereafter adding aqueous phosphoric acid tomixture to adjust the pH of the aqueousphase to a'value between about1.9 and 3.0, separating and discarding the aqueous 7 phase, treating theethylene dichloride extract containing unadsorbcd' penicillin from thecharcoal and impurities. 1

10. A process for obtaining penicillin from a slightly acidic aqueousextract of a penicillinproducing mold culture, which comprises chillingsaid aqueous'extract to produce solid impurities therein, separatingsaid impurities, extracting further impurities-from the aqueous residueat approximately neutral pH with a water-immiscible organicsolventfor'oily impurities, adding an organic solventto the extracted aqueousfurther impurities from the aqueous residue at I approximately neutralpH with ether, adding residue, quickly thereafter adding aqueous mineralacid to mixture toadjust the pH- of the aqueous phase to a value betweenabout 1.9 and .resldue, quickly thereafter adding aqueous min- 3.0,separating and discarding the aqueous phase,

treating the organic solvent extract with activated charcoal to adsorbimpurities upon said charcoal, and separating the organic extract con- Itaining unadsorbed penicillin from the charcoal and impurities.

11. The process for purifying partially purified penicillin material,which comprises adding to a solution or the penicillin in a non-polarwater immiscible solvent, a relatively small proportion of'surtacc-activecarbon,thereby adsorbing the impurities withoutsubstantial adsorbtion of the penicillin, and then separating thecarbon, with T impurities adsorbed, from the purified penicillinsolution.

12. A- process for'obtaining penicillin from a chloroform extractcontaining the same, which coal and impurities.

containing comprises treating said extract with activated.

.. j charcoal, and'separating the chloroform'extract containingunadsorbed penicillin from the char-- 1s. A process for'obtaining'penicillin from an am'yl acetate extract containing the same, whichcomprises treatingjsaid -extract with activated charcoal, and separatingthe-amyl acetate extract coal and impurities.

1 ounu'rmnnaarz.

emce crran The following 'reierences. are'ot record in the flleoi thispatent=- Lancet, August 16, 1941, pages 177-189. British Journal orExperimental Pathology, vol. 23, No. e, JunelMZpages 103-122.

Manufacturing Chemist & Manufacturing Periumer. August1943, XIV. mes251-254.

Charles Pflzen-Beport No. 1, January 2, 1% (pages land 22).

Chem. 8: Met. Engineering. April 1944, Artice with activated charcoal toadsorb impurities upon b c. mum 10 m mu said charcoal, and separatingthe organic extract penicillin from the charg

11. THE PROCESS FOR PURIFYING PARTIALLY PURIFIED PENICILLIN MATERIAL,WHICH COMPRISES ADDING TO A SOLUTION OF THE PENICILLIN IN A NON-POLARWATER IMMISCIBLE SOLVENT, A RELATIVELY SMALL PROPORTION OFSURFACE-ACTIVE CARBON, THEREBY ADSORBING THE IMPURITIES WITHOUTSUBSTANTIAL ADSORBTION OF THE PENICILLIN, AND THEN SEPARATING THECARBON, WITH IMPURITIES ADSORBED, FROM THE PURIFIED PENICILLIN SOLUTION.